GOFAST: an integrated approach for efficient and comprehensive membrane proteome analysis.
Anal Chem
; 84(21): 9008-14, 2012 Nov 06.
Article
en En
| MEDLINE
| ID: mdl-23030679
ABSTRACT
Membrane proteomics, the large-scale analysis of membrane proteins, is often constrained by the difficulties of achieving fully resolvable separation and resistance to proteolysis, both of which could lead to low recovery and low identification rates of membrane proteins. Here, we introduce a novel integrated approach, GELFrEE Optimized FASP Technology (GOFAST) for large-scale and comprehensive membrane proteins analysis. Using an array of sample preparation techniques including gel-eluted liquid fraction entrapment electrophoresis (GELFrEE), filter-aided sample preparation (FASP), and microwave-assisted on-filter enzymatic digestion, we identified 2 090 proteins from the membrane fraction of a leukemia cell line (K562). Of these, 37% are annotated as membrane proteins according to gene ontology analysis, resulting in the largest membrane proteome of leukemia cells reported to date. Our approach combines the advantages of GELFrEE high-loading capacity, gel-free separation, efficient depletion of detergents, and microwave-assisted on-filter digestion, minimizing sample losses and maximizing MS-detectable sequence coverage of individual proteins. In addition, this approach also shows great potential for the identification of alternative splicing products.
Texto completo:
1
Bases de datos:
MEDLINE
Asunto principal:
Proteoma
/
Proteómica
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Electroforesis
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Métodos Analíticos de la Preparación de la Muestra
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Proteínas de la Membrana
Límite:
Humans
Idioma:
En
Revista:
Anal Chem
Año:
2012
Tipo del documento:
Article
País de afiliación:
Estados Unidos